Cyclosporin A (CsA) and FK506 are widely used immunosuppressive drugs that inhibit the Ca2+-activated phosphatase calcineurin. Calcineurin's major activity in T cells is thought to be dephosphorylation (and thus activation) of the NFAT family of transcription factors, which are important for cytokine production and cell proliferation. It inhibition of this process that is thought to underlie the therapeutic efficacy of CsA in the treatment of transplant rejection and autoimmune diseases. It has been reported that CsA also inhibits the classic MAP kinase cascade, and therefore the stress-induced activation of p38. Signaling via the TCR results in the activation of the MAP kinase p38 by a MAPK cascade-independent mechanism: phosphorylation of p38 Y323 by ZAP-70. Therefore, we were surprised to observe that CsA also inhibited p38 activation downstream of TCR signaling. This led us to explore the molecular mechanism for this inhibition, and we have made the following observations: 1. Remarkably, CsA and FK506 inhibited a number of key events in proximal T cell signaling. These include phosphorylation of ZAP-70 on Y493, LAT, and SLP76, whereas phosphorylation of ZAP-70 Y319, PLCgamma1, and ERK were intact. 2. Calcineurin itself is recruited to the TCR signalosome within two minutes of activation and then slowly dissociates over the next 30-60 minutes. This was confirmed both by co-immunoprecitation studies as well as imaging of TCR micro clusters by confocal microscopy. 3. Calcineurin recruitment requires Lck signaling and ZAP-70, but not ZAP-70 signaling. Therefore, we believe that it is binding phosphorylated ZAP-70. 4. siRNA-mediated knockdown of calcineurin recapitulates inhibition of ZAP-70 493, and introduction of ZAP-70 Y493R (which cannot be phosphorylated on residue 493) results in signaling defects similar to those described above (point 1). 5. The calcineurin target in the TCR signalosome is Lck S59, whose phosphorylation has been reported to inhibit TCR signaling. Consistent with this, introduction of Lck mutants (Lck S59A, which can not be phosphorylated, and Lck S59E, a phospho-mimetic) resulted in enhanced and inhibited signaling, respectively. 6. To determine if inhibition of proximal TCR signaling could be a novel mechanism for it's immunosuppressive effects, we looked for important NFAT-independent events downstream of TCR signaling. LFA-1-dependent adhesion to ICAM-1 is a very rapid and essential step in T cell activation and egress from the blood to tissue. We found that CsA inhibited conversion of LFA-1 from the inactive to the active state and blocked cell adhesion to ICAM-1-coated wells. Studies with T cells expressing the Lck-mutants confirmed that the phosphorylation status of S59 was responsible. Followup studies have shown that the addition of CsA to T cells already activated and bound to ICAM-1-coated plates or antigen-pulsed APC results in rapid (minutes) reversal of adhesion. These results have shown that, unexpectedly, calcineurin is an integral member of the TCR signaling complex. Moreover, it's inhibition by immunosuppressive drugs has important effects on T cell adhesion, which can account for at least some of the therapeutic efficacy of this class of reagents. Studies on the possible function of calcineurin as an adaptor protein in the signaling complex are ongoing. In a separate study we have found that p38, which is activated by the tyrosine kinase ZAP-70, in turns phosphorylated ZAP-70 on a novel site, T293. This phosphorylation destabilizes the signaling complex, resulting in attenuation of signaling. When this Thr is replaced with an Ala, TCR signaling is prolonged, resulting in enhanced effector functions. Therefore, there is a tight negative feedback loop involving two kinases that are part of the proximal TCR signaling machinery.